Gluten intake and gluten-free diet in the Netherlands Hopman, G.D.

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Gluten intake and gluten-free diet in the Netherlands

Hopman, G.D.

Citation

Hopman, G. D. (2008, September 25). Gluten intake and gluten-free diet in the Netherlands.

Retrieved from https://hdl.handle.net/1887/13118

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden

Downloaded from: https://hdl.handle.net/1887/13118

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Gluten intake and gluten-free diet in the Netherlands

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Cover illustration gluten structure obtained by washing out dough.

Cover design Anja Bakker

Printed by PrintPartners Ipskamp B.V., Enschede, The Netherlands ISBN 978-90-9023381-9

Financial support for the publication of this thesis was kindly provided by Willem- Alexander Kinder- en Jeugdcentrum (Leiden University Medical Center), Celiac Disease Consortium, Dienst Diëtetiek (Leiden University Medical Center), Divisiebestuur Divisie 2 (Leiden University Medical Center), Nederlandse Coeliakie Vereniging, Friso

Kindervoeding, Mead Johnson Nutritionals, Nutricia Advanced Medical Nutrition, Sorgente B.V., Nutri-akt b.v. - arbeidsmarktintermediair binnen Food, Technology, Nutrition & Health.

No part of this thesis may be reproduced in any form without written permission from the author.

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Gluten intake and gluten-free diet in the Netherlands

Proefschrift

ter verkrijging van

de graad van Doctor aan de Universiteit Leiden,

op gezag van Rector Magnificus prof.mr. P.F. van der Heijden, volgens besluit van het College van Promoties

te verdedigen op donderdag 25 september 2008 klokke 15.00 uur

door

Geertruida Dorothea Hopman

geboren te Den Burg, Texel in 1965

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Promotiecommisie:

Promotor: Prof. dr. J.M. Wit

Copromotor: Dr. M.L. Mearin-Manrique

Referent: Prof. dr. C.J.J. Mulder (VU Amsterdam)

Overige leden: Dr. L.J.W.J. Gilissen (Wageningen Universiteit, Wageningen) Prof. dr. D.W. Hommes

Prof. dr. J.A. Romijn

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General introduction, aim and outline of this thesis

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In the early 1930’s the Dutch paediatrician W.K. Dicke discovered that the elimination of wheat from the diet was beneficial for celiac patients (1). This benefit was confirmed later during the period of food shortage in the Second World War (1944-1945), during which bread was unavailable, and Dicke observed that the clinical condition of the hospitalized children with celiac disease improved. Further studies showed that gluten and specifically its alcohol-soluble component, gliadin, was harmful for celiac patients (2). Gluten is the storage protein of wheat and wheat-related grains and can be subdivided in the gliadin and glutenin protein families, both of which are involved in celiac disease. Since then, a gluten-free diet has been the basis of the treatment of celiac patients.

In celiac patients, gluten causes histological alterations of the small bowel that may lead to disturbances in nutrient absorption and symptoms such as diarrhea, failure to thrive, abdominal pain, and extraintestinal complications such as osteoporosis, infertility and cancer (3). The treatment of celiac disease consists of a life-long gluten-free diet to heal the duodenal mucosa, improve symptoms, and protect against development of

complications (4,5).

The diagnosis of celiac disease is based on characteristic histological alterations of the small bowel mucosa during gluten consumption and clear clinical remission with a gluten-free diet. In asymptomatic patients, however, a control biopsy is needed to prove mucosal recovery after treatment (6). In earlier days, a third biopsy after gluten challenge was needed to confirm the diagnosis in children (7).

Celiac disease is considered to be a life-long disorder, but there are studies describing patients diagnosed with celiac disease in childhood who seem to tolerate gluten later in life for an extended period of time (8).

Until now, the only effective treatment for the disease has consisted of a gluten-free diet in which wheat, rye, barley, spelt, kamut, and products derived from these cereals are avoided. To what extent oats belong to the list of banned cereals is still debated. Both long-term follow-up studies (9-11) and laboratory studies on oats being less toxic than wheat (12), support that oats are permitted in the gluten-free diet for adults and children.

However, some patients do show mucosal damage after oat consumption, and individual differences in oat tolerance have been found (11,13). In the Netherlands, as in many other countries, fear of wheat contamination in commercially available oat products has led to a reluctance to recommend oats to celiac patients (14,15).

Wheat cereal is a staple food in many countries in Europe and is widely used in the food industry. Therefore, wheat is difficult for celiac patients to avoid; hence, the prescription to follow a gluten-free diet has a big impact on the patients’ daily and social life (16), and even on the lives of family members. The availability of the gluten-free products is

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limited, and consequently, celiac patients have difficulty finding gluten-free foods.

Furthermore, taste as well as higher expenses can be limiting factors for compliance. For example, in the Netherlands, only a few health insurance companies contribute to the added costs of the gluten-free products. All these factors may affect the health-related quality of life of celiac patients (17,18). Furthermore, the nutritional value of gluten-free food products is lower compared to the gluten-containing equivalents, which may lead to inadequate nutrient intake (19-22).

Genetics play a role in the development of the disease: as much as 98% of the celiac patients are HLA-DQ2 (95%) or –DQ8 (3%) positive. However, the majority of people with these genetic factors do not develop celiac disease. This suggests that additional genetic and/or environmental factors play a role in disease development.

Many genetic and immunological studies have been performed in an attempt to unravel the complexity of this multi-factorial disease (23-25). In addition, the possible role of environmental factors, such as early feeding, in the development or prevention of celiac disease has been studied (26-28). The Swedish ‘experiment of nature’ causing the rise and fall of ‘an epidemic’ of gluten intolerance after changes in infant feeding suggests that early feeding may be an important factor (26). Breastfeeding at the time of gluten introduction, ongoing breastfeeding while gluten is already being consumed, as well as timing and amount of gluten introduced into the diet, may play a preventive role in the development of celiac disease (27-29).

Breastfeeding and weaning influence the development of the gastro-intestinal tract, and it is possible that gradual introduction of antigens will lead to the development of oral tolerance (30,31). It is also likely that the response of the immune system to gluten is modified by breastfeeding (32,33). The presence of gluten peptides in breast milk leading to an early exposure to gluten, even before gluten is introduced into the infants’ diet, has been studied as a possible factor in the development of oral tolerance (34,35).

AIMS OF THIS THESIS

Gluten is essential for the development of celiac disease: in the absence of gluten, celiac disease will not be expressed. The aims of this thesis were to explore the relationship of celiac patients with gluten and the gluten-free diet at different ages, their ability to develop tolerance to gluten, and the impact of the gluten-free diet on health-related quality of life. Furthermore, this thesis also aims to measure some of the environmental factors such as breastfeeding and gluten intake in early life considered to play a role in the prevention of celiac disease and in the possible development of oral tolerance.

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Chapter 1 consists of a general introduction and description of the aims and outline of the thesis. In chapter 2, the implication of the presence of gluten proteins in breast milk for the development of celiac disease is discussed. This relates to one of the aims of the study, i.e. to measure some of the environmental factors possibly involved in celiac disease, such as early feeding. Breastfeeding has been shown to prevent, or at least delay, the development of celiac disease (28). Breast milk contains many immunological factors that stimulate the infant’s immune system, but its exact role in the prevention of celiac disease is not known. Furthermore, breast milk contains small amounts of food antigens, like gluten peptides, that may contribute to tolerance induction. As the first contact with gluten may be important in this respect and the level of gluten peptides in breast milk may vary with intake of gluten by the mother, we studied the level of gluten peptides in breast milk of mothers on a gluten-containing diet and of mothers on a gluten-free diet.

Expecting to find gluten peptides in the breast milk of mothers on a normal diet, but not in the breast milk of mothers on a gluten-free diet.

In chapter 3 we describe the development and testing of a food questionnaire to assess gluten intake, since another possible factor in the development of oral tolerance is the timing of gluten introduction and the quantity of gluten consumption in early life.

However, this hypothesis is only based on observational studies. The role of gluten introduction should be confirmed by intervention studies before cause-effect conclusions can be drawn and changes in advice considering early infant feeding can be proposed.

For such studies, it would be necessary to have an instrument available to assess the amount of gluten consumed. Such an instrument should be accurate, easy to use, and should be easily accessible by both researchers and parents. Since such an instrument was lacking, we developed and validated one for this purpose.

In exploring the attitude of celiac patients towards the gluten-free diet, we studied the management of the gluten-free diet by adolescent celiac patients. The results of that study are described in chapter 4. Until now, the gluten-free diet was the only effective

treatment for the disease. From the perspective of the celiac patient, this treatment is quite a burden. Gluten intake is difficult to avoid since wheat is the cereal most used in staple food and widely used in the food industry. Furthermore, adherence to a gluten-free diet may have negative nutritional consequences. Dietary compliance in adolescents with celiac disease has been studied frequently and was shown to vary between 52% and 81%

in European countries. In the Netherlands, however, we did not have information on this topic. Therefore, we studied the situation of dietary compliance in our country and the consequences for the nutrient intake in young celiac patients.

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In an attempt to enlarge the gluten-free food choices, we studied whether the (new) naturally gluten-free cereal, tef (Eragrostis tef) can be safely used by celiac patients (Chapter 5). Adherence to the diet is often reported as being difficult. Next to the aforementioned aspects, the limited availability, the variety and the taste of gluten-free food products may have negative effects on the compliance with the gluten-free diet. A greater variety of tasteful products may contribute to a better compliance with the diet.

We studied the health-related quality of life (Chapter 6) in an adult population of celiac patients. Having a chronic disorder as well as having to adhere to a dietary regimen may affect quality of life. The health-related quality of life of celiac patients adhering to the gluten-free diet has been frequently studied in children as well as in adults. The adult population we studied was recruited for the study described in chapter 7 and consisted of celiac patients with strict adherence to the gluten-free diet and of celiac patients with gluten transgression or consuming a normal gluten-containing diet. This gave us the opportunity to compare the results of the health-related quality of life survey between compliers and non-compliers.

In chapter 7 we describe the results of a study on possible development of tolerance to gluten. As celiac disease is considered to be a permanent disorder, the diet has to be followed for life. However, patients consuming gluten without developing symptoms or signs of the disease have been described. Therefore, it is important to investigate which factors (genetic, immunologic or environmental) determine which patients with celiac disease remain intolerant to gluten for life and which few may regain tolerance. We studied the possible existence of adult celiac patients developing tolerance to gluten in the Netherlands and whether we could identify immunological or genetic factors that might contribute to this.

Finally, the overall results of the studies described in this thesis are discussed in chapter 8.

REFERENCES

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2. Kamer JH van de, Weijers HA, Dicke WK. Coeliac disease. IV. An investigation into the injurious constituents of wheat in connection with their action on patients with coeliac disease. Acta Paediatr 1953;42:223-31.

3. Green PHR, Jabri B. Coeliac disease. Lancet 2003;362:383-91.

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4. Mora S, Weber G, Barera G, Bellini A, Pasolini D, Prinster C, Bianchi C, Chiumello G.

Effect of gluten-free diet on bone mineral content in growing patients with celiac disease. Am J Clin Nutr 1993;57:224-30.

5. Molteni N, Bardella T, Bianchi PA. Obstetric and gynecological problems in women with untreated coeliac sprue. J Clin Gastroenterol 1990;12:37-9.

6. Walker-Smith JA, Guandalini S, Schmitz J, Shmerling DH, Visakorpi JK. Revised criteria for the diagnosis of coeliac disease. Report Working Group European Society of Paediatric Gastroenterology and Nutrition. Arch Dis Child 1990;65:909-11.

7. Meeuwisse GW. Diagnostic criteria in coeliac disease. Acta Paediatr Scand 1970;59:461- 3.

8. Matysiak-Budnik T, Malamut G, Patey-Mariaud de Serre N, Grosdidier E, Seguier S, Brousse N, Caillat-Zucman S, Cerf-Bensussan N, Schmitz J, Cellier C. Long-term follow- up of 61 celiac patients diagnosed in childhood: evolution toward latency is possible on a normal diet. Gut online publication February 15, 2007 as 10.1136/gut.2006.100511.

9. Janatuinen EK, Kemppainen TA, Julkunen RJK, Kosma VM, Maki M, Heikkinen M, Uusitupa MI. No harm from five year ingestion of oats in coeliac disease. Gut 2002;50:

332-5.

10. Holm K, Mäki M, Vuolteenaho N, Mustalahti K, Ashorn M, Ruuska T, Kaukinen K.

Oats in the treatment of childhood coeliac disease: a 2-year controlled trial and long-term clinical follow-up study. Aliment Pharmacol Ther 2006;23:1463-72.

11. Garsed K, Scott BB. Can oats be taken in a gluten-free diet? A systematic review. Scand J Gastroenterol 2007;42:171-8.

12. Vader LW, Stepniak DT, Bunnik EM, Kooy YMC, de Haan W, Drijfhout JW, van Veelen P, Koning F. Characterization of cereal toxicity for celiac disease patients based on protein homology in grains. Gastroenterology 2003;125:1105-13.

13. Lundin KE, Nilsen EM, Scott HG, Loberg EM, Gjoen A, Bratlie J, Skar V, Mendez E, Lovik A, Kett K. Oats induced villous atrophy in coeliac disease. Gut 2003;52:1649-52.

14. Hernando A, Mujico JR, Juanas D, Mendez E. Confirmation of the cereal type in oat products highly contaminated with gluten. J Am Diet Assoc 2006;106:665-6.

15. Thompson T. Gluten contamination of commercial oat products in the United States. N Engl J Med 2004;351:2021-2.

16. Hallert C, Grännö C, Grant C, Hultén S, Midhagen G, Ström M, Svensson H, Valdimarsson T, Wickström T. Quality of life of adult coeliac patients treated for 10 years. Scand J Gastroenterol 1998;33:933-8.

17. Zarkadas M, Cranney A, Case S, Molloy M, Switzer C, Graham ID, Butzner JD, Rashid M, Warren RE, Burrows V. The impact of a gluten-free diet on adults with celiac disease:

results of a national survey. J Hum Nutr Dietet 2006;19:41-9.

18. van Doorn RK, Winkler LMF, Zwinderman KH, Mearin ML, Koopman HM. The CDDUX: A disease-specific health-related quality-of-life questionnaire for children with celiac disease. In press.

19. Mariani P, Viti MG, Montuori M, La Vecchia A, Cipolletta E, Calvani L, Bonamico M.

The gluten-free diet: a nutritional risk for adolescents with celiac disease. J Pediatr Gastroenterol Nutr 1998;27:519-23.

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20. Hallert C, Grant C, Grehn S, Grännö C, Hultén S, Midhagen G, Ström M, Svensson H, Valdimarsson T. Evidence of poor vitamin status in coeliac patients on a gluten-free diet for 10 years. Aliment Pharmacol Ther 2002;16:1333-9.

21. Thompson T. Thiamin, riboflavin, and niacin contents of the gluten-free diet: Is there cause for concern? J Am Diet Assoc 1999;99:858-62.

22. Thompson T. Folate, iron, and dietary fiber contents of the gluten-free diet. J Am Diet Assoc 2000;100:1389-96.

23. Monsuur AJ, Wijmenga C. Understanding the molecular basis of celiac disease: What genetic studies reveal. Ann Med 2006:38:578-91.

24. Koning F. The molecular basis of celiac disease. J Mol Recognit 2003;16:333-6.

25. Stepniak D, Koning F. Celiac disease – sandwiched between innate and adaptive immunity. Human Immunology 2006;67:460-8.

26. Ivarsson A, Persson LÅ, Nyström L, Ascher H, Cavell B, Danielsson L, Dannaeus A, Lindberg T, Lindquist B, Stenhammar L, Hernell O. Epidemic of celiac disease in Swedish children. Acta Paediatr 2000;89:65-71.

27. Ivarsson A, Hernell O, Stenlund H, Persson LÅ. Breast-feeding protects against celiac disease. Am J Clin Nutr 2002;75:914-21.

28. Akobeng AK, Ramanan AV, Buchan I, Heller RF. Effect of breast feeding on risk of coeliac disease: a systematic review and meta-analysis of observational studies. Arch Dis Child 2006;91:39-43.

29. Norris JM, Barriga K, Hoffenberg EJ, Taki I, Miao D, Haas JE, Emery LM, Sokol RJ, Erlich HA, Eisenbarth GS, Rewers M. Risk of celiac disease autoimmunity and timing of gluten introduction in the diet of infants at increased risk of disease. JAMA

2005;293:2343-51.

30. Brandtzaeg PE. Current understanding of gastrointestinal immunoregulation and its relation to food allergy. Ann NY Acad Sci 2002;964:13-45.

31. Strobel S. Oral tolerance, systemic immunoregulation and autoimmunity. Ann NY Acad Sci 2002;968:47-58.

32. Falth-Magnusson, Franzen L, Jansson G, Laurin P, Stemhammer L. Infant feeding history shows distinct differences between Swedish celiac and reference children. Pediatr Allergy Immunol 1996;7:1-5.

33. Hanson LA. Breastfeeding provides passive and likely long-lasting active immunity. Ann Allergy Asthma and Immunol 1998;8:523-37.

34. Chirdo FG, Rumbo M, Anon MC, Fossati CA. Presence of high levels of non-degraded gliadin in breast milk from healthy mothers. Scand J Gastroenterol 1998;33:1186-92.

35. Troncone R, Scarcella A, Donatiello A, Cannataro P, Tarabuso A, Auricchio S. Passage of gliadin into human breast milk. Acta Paediatr Scand 1987;76:453-6.

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CHAPTER 2

Presence of gluten proteins in breast milk:

implications for the development of celiac disease

Erica Hopman^#, Liesbeth Dekking^#, Femke Beelen, Natascha Smoltsak, Sandra de Vries, Lineke Dogger, Luisa Mearin and Frits Koning

#Both authors contributed equally to the work described in this paper

This study was supported by the Celiac Disease Consortium, an Innovative Cluster approved by the Netherlands Genomics Initiative and partially funded by the Dutch Government (BSIK03009)

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ABSTRACT

Aims: Celiac disease (CD) is a multifactorial disease with a strong genetic association and is caused by a T cell mediated immune response to gluten. Although much is known about the molecular mechanism, the trigger for the onset of CD is still unclear. A correlation between a reduced risk of developing CD and breastfeeding has been shown.

The preventive mechanism however, remains unclear. In this study we test the

hypothesis that T cell stimulatory epitopes originating from dietary gluten appear in the human breast milk.

Method: Breast milk of 23 mothers on a normal diet (mean gluten intake: 17 g/day) and 13 mothers on a strict gluten-free diet was collected in the period from one week until eight months after delivery. The presence of gluten proteins was studied using monoclonal antibody based competition assays specific for the T cell stimulatory epitopes of gliadin (Glia-9, Glia-20, Glia-γ1) and glutenin (Glt-156 and High Molecular Weight (HMW).

Results: T cell stimulatory epitopes of both gliadin and glutenin (Glia-α9 and HMW glutenin) were detected in the breast milk of mothers on a normal diet. Correlation studies revealed that the gluten intake was not correlated with the level of the Glia-α9 T cell stimulatory epitope detected.

Conclusion: Infants are exposed to small levels of gluten via breast milk. It is tempting to assume that these low levels of gluten are responsible for the induction of oral tolerance to gluten.

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INTRODUCTION

Celiac disease (CD) is an inflammatory intestinal disorder caused by immune responses induced by dietary gluten proteins (1-3). It is a multi factorial disease with a strong HLA association. Approximately 95% of celiac disease patients are HLA-DQ2 (α1*0501, β1*0202), whereas the remainder is usually HLA-DQ8 (α1*0301, β1*0302).

With a prevalence of approximately 1 in 100-200, CD is the most common food induced enteropathy in the western world. A wide range of variable symptoms are associated with CD, including abdominal pain, diarrhea, constipation, vomiting, osteoporosis, growth retardation, and migraine. Many patients, however, have only very mild or no apparent clinical symptoms and are never diagnosed properly. At present, the only possible treatment for CD patients is a strict life-long gluten-free diet (GFD).

Although feasible, a GFD is complicated by the widespread use of gluten in the food industry and as an additive to many products that are not normally associated with gluten or wheat, like medication. Moreover, a strict GFD causes a severe restriction in the patients’ social life (4).

Gluten molecules are the storage proteins of wheat and can be subdivided in the gliadin and glutenin protein families, both involved in CD. The proteins have a high proline content (5) and as a result, gluten proteins are poorly degraded by enzymes of the gastrointestinal tract (6). In the small intestine of CD patients, the partially degraded gluten proteins are modified by the activity of the enzyme tissue transglutaminase (tTG) (7-9). This so called deamidation introduces a negative charge in gluten peptides which facilitates their binding to the disease predisposing HLA-DQ2/8 molecules and facilitates efficient presentation of gluten peptides to CD4+ T cells of the immune system (7-14).

The T cell response against gluten is specific for CD patients since no evidence of T cell mediated reactivity against dietary gluten has been reported in normal, non-celiac, mucosa. The gluten reactive T cells have a Th0/Th1 phenotype and usually release the proinflammatory cytokine IFN-γ (15). Although at least 50 T cell stimulatory epitopes in gluten proteins have been identified, a unique 33-mer peptide of -gliadin seems to be the most immunogenic (6,16). This 33-mer harbours six in part overlapping epitopes and it is resistant to the enzymatic degradation by gastric, pancreatic and brush border enzymes.

Although much is known about the molecular mechanism underlying CD, little is known about the onset and possibilities to prevent the disease. Since only a minority of the genetically predisposed individuals actually develop CD, a threshold of tolerance was suggested (17). This threshold is influenced by both gene dose (18) and gluten exposure (19). A large repertoire of abundant immunogenic gluten peptides in the diet, together with a high copy number of HLA-DQ2, thus may favor the breaking of oral tolerance. In

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(20). As there is no restriction in the amount of gluten given, gluten intake at the age of 12 months is between 6 and 9 grams daily (21), while gluten-specific T cells of CD patients are known to respond to microgram amounts. The sudden introduction of grams of gluten may thus play an important role in the breaking of oral tolerance.

Another factor influencing the threshold or breaking of tolerance is breastfeeding.

Recent studies have shown that breastfeeding offers protection against the development of CD (19,22). Breastfeeding at the time of gluten introduction and ongoing

breastfeeding while gluten is already being consumed were associated with a reduced risk of development of CD. The exact preventive mechanism of breastfeeding on the development of CD, however, remains unclear. A tentative explanation might be that small amounts of gluten in breast milk promote the induction of low dose oral tolerance against gluten.

In this study, the presence of gluten in breast milk of mothers on a normal gluten- containing diet (ND), was investigated and compared to a control group of mothers on a GFD.

MATERIALS AND METHODS Subjects

In 2005 lactating women on a ND were contacted at random at one Child Health Care Center (Nieuw Vennep, the Netherlands). In the year 2004 year, the Child Health Care Centers were attended by 91% of the families with infants in the Netherlands (23).

Lactating mothers on a GFD were contacted through a call in ‘Glutenvrij’ a periodical distributed among members of the Dutch Celiac Disease Society (NCV), and a call on the website of the NCV.

Samples

Breast milk of women on a ND and of women on a GFD was collected longitudinally.

Both groups of participants were asked to collect three samples of breast milk (morning, afternoon and evening) once a month. The samples were stored at home in labeled tubes in the freezer. After collection, the samples were transported to the laboratory, thawed, subdivided into small portions and stored at -70°C. Before analysis, one portion of each sample was thawed and the whey fraction was obtained by centrifugation at 14,000 rpm for 30 minutes at room temperature, after which the fat was removed.

Competition assays for the quantitative detection of T cell stimulatory epitopes of gluten proteins

Competition assays were performed as described earlier (24-26). For quantification of the gliadin assays, a standard curve was made by the Prolamine Working Group gliadin standard (27) in a concentration range of 10 μg/ml-10 ng/ml. The assays specific for the

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detection of T cell stimulatory epitopes of LMW glutenin were calibrated using a 25-mer synthetic peptide as a standard that contains the Glt-156 epitope (14). The HMW- glutenin specific assay was calibrated using a chymotrypsin digest of purified HMW- glutenin proteins (kindly provided by P. Shewry, Rothamsted Research, Hampenden, United Kingdom). Both glutenin standards were used in a concentration range from 1

g/ml-2 ng/ml.

Food record and gluten calculation

The mothers on a ND recorded a food record on three consecutive days preceding the day of breast milk collection. The last day of recording coincided with the day of breast milk collection. The amounts of food consumed were recorded in household measures and the name of the manufacturer of the products used was precisely written down. To determine the gluten intake, the vegetable protein content of the gluten-containing products was calculated according to the Dutch Food Composition Table (28). Since there are no analyses on gluten content of products, this was calculated by multiplying the grams of vegetable protein of the gluten-containing food by 0.8 as described by Overbeek et al., (29) and by linking a food composition table spreadsheet to food

consumption data using MS Access 2000. The products that may contain gluten, but with missing brand information or with a rounded number of zero grams protein in the food composition table, were defined by us as 'risk products'. As an assumption for the gluten content in those risk products an amount of 20 mg gluten per 100 g food product was used, which is the maximum of the Codex Alimentarius norm (30) for gluten-free products.

Data analysis

The data obtained by the competition assays and the commercial gliadin ELISA were imported in the scientific graphing and statistics program Graph Pad Prism version 4.02 (GraphPad Software, Inc. San Diego CA, USA). The significance of the differences detected between the level of gluten epitopes in the breast milk of mothers on a ND vs those on a GFD were assessed with a 2-sided unpaired t-test. P<0.05 was considered to indicate a significant difference. The Glia-9 epitope is part of the degradation resistant 33-mer of alpha gliadin (16), and therefore the most likely to be detected in the breast milk as compared to Glia-20, Glia-1 and HMW glt. Correlations between grams of gluten intake of the mother and Glia-9 concentrations in breast milk were carried out using SPSS version 14.0 for Windows and checked by the Pearson correlation test.

P<0.05 was considered to indicate a significant correlation.

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RESULTS Subjects

Twenty-three mothers on a ND (mean age 33±5 y) and 13 mothers on a GFD (mean age 34±4 y) responded and joined the study. Twelve of the 13 mothers on a GFD were CD patients diagnosed by small bowel biopsy. One mother showed clinical symptoms of CD and went on a GFD without being diagnosed.

Figure 1:Distribution of breast milk samples collected at different months of lactation.

Breast milk samples were collected longitudinally at different stages of lactation of mothers on a normal diet (ND, n=131) and mothers on a gluten-free diet (GFD, n=116). In both groups, most samples were obtained from month 2 and 3 of the lactation period.

Breast milk samples

Breast milk samples were collected from the first week after delivery up to eight months of lactation. A total of 131 samples of mothers on a ND and 116 samples of mothers on a GFD were obtained. The sample distribution over the various months of lactation was comparable for both groups with the highest number of samples obtained in months 2 and 3 of lactation (Figure 1).

The presence of gliadin- and glutenin-derived T cell stimulatory epitopes in breast milk

The presence of T cell stimulatory epitopes of gluten proteins known to be involved in CD (both from gliadin and glutenin) was determined in the whey fractions of the samples using mAb-based competition assays. In both groups of samples, ND vs GFD, T cell

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stimulatory epitopes were detected in the assays specific for the Glia-9, Glia-20, Glia-

1 and HMW-glutenin T cell stimulatory epitopes (Figure 2).

Figure 2:Level of T cell stimulatory epitopes of gliadin and glutenin in breast milk.

Whey fraction of breast milk of mothers on a normal diet (ND) and mothers on a gluten-free diet (GFD) were measured in the mAb-based competition assays specific for T cell stimulatory epitopes of gliadin and glutenin. Mean level per mother of (A) the Glia-9, (B) the Glia-20, (C) the Glia-γ1 and (D) the HMW glutenin-derived T cell stimulatory epitope.

For the LMW glutenin-derived T cell stimulatory epitopes no results were obtained since the LMW specific competition assay was not suited to measure the presence of the T cell stimulatory epitopes in breast milk. T cell stimulatory epitopes Glia-9 and HMW glutenin present in the samples from mothers on a ND were significantly increased (P<0.05) compared to the levels in the samples of mothers on a GFD. The differences between the levels of the T cell stimulatory Glia-α20 and Glia-γ1 epitopes were higher in women on a ND, but did not reach significance (Table 1).

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Table 1:Levels of T cell stimulatory epitopes of gliadin and glutenin in breast milk of mothers on a normal diet versus mothers on a gluten-free diet.

Normal diet (n=131) Gluten-free diet (n=116) Epitope

mean SEM mean SEM P

Glia-9 (g/ml) 0.1324 0.0187 0.0615 0.0088 0.0135

Glia-20 (g/ml) 0.1285 0.0425 0.0549 0.0181 0.5410

Glia-1 (g/ml) 0.4038 0.0471 0.3669 0.0394 0.7922

HMW glt (ng/ml) 44.92 6.388 22.92 6.162 0.0351

SEM = standard error of the mean; p indicates a two sided p value for the difference between gliadin and glutenin levels detected in breast milk of mothers on a normal diet compared to mothers on a gluten-free diet (unpaired t test); P<0.05 indicate a significant difference between the compared mean values.

Gluten intake of mothers on a normal, gluten-containing diet

Of the 23 mothers on a ND 22 kept a 3-day food record preceding the day of breast milk collection. In total, 48 food records were collected and the mean gluten intake was 17±3.8 g/day. The median consumption of gluten from risk products was 1.5 mg/day which is less than 0.01% of the total gluten intake. Because of this small percentage, the gluten intake from risk products was not taken into account in further analysis.

No correlation between gluten intake and level of Glia-9 detected in breast milk of mothers on a normal diet

The correlation between gluten intake and the level of gluten epitopes detected in the breast milk of mothers on a ND was studied using the mean levels detected for the degradation resistant Glia-α9 epitope. This epitope is part of the degradation resistant 33- mer of alpha gliadin (16), and therefore the most likely to be detected in the breast milk.

We checked the correlation between the gluten intake of the mothers on a ND and the level of Glia-9. No correlations were found between the mean level of Glia-α9 epitope detected and the amount of gluten consumed in the three or two days preceding the day of breast milk collection (r= -0.142, P=0.37; r= -0.131, P=0.42, respectively). In addition, no correlation was detected between the mean level of the Glia-α9 epitope and the gluten intake during day 2 or day 3 (r= -0.104, P=0.52; r= -0.109, P=0.50, respectively).

DISCUSSION

In this study, breast milk samples were analyzed by antibody based assays specific for the detection of T cell stimulatory gluten peptides, for the presence of dietary gluten-derived peptides involved in the development of CD. Increased levels of peptides of both gliadin and glutenin could be shown in breast milk of mothers on a ND compared to those in

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the breast milk of mothers on a GFD. The detected level of Glia-9 however, did not correlate with the gluten intake of the mothers on a ND. Our result indicates that breast- fed children are exposed to low amounts of gluten-derived T cell stimulatory peptides involved in CD, even before gluten is introduced into the infants’ diet.

The presence of gluten proteins in breast milk has been reported previously (31,32). The mean level of gliadin in breast milk of 178 ng/ml (range: 5-2000 ng/ml) reported in previous studies (31,32) correlates well with the mean levels of T cell stimulatory epitopes of gliadin detected in our study (Glia-9 mean level 132 ng/ml, range 20.2-392 ng/ml;

Glia-20 mean level 129 ng/ml, range 0-737 ng/ml and Glia-1 mean level 404 ng/ml, range 143.7-1059 ng/ml).

Moreover, with the recently developed competition assay specific for the T cell stimulatory epitope of HMW glutenin (25), we could show that next to gliadin HMW glutenin also appears in breast milk (HMW mean level 44.92 ng/ml, range 2.23-104.8 ng/ml). Regarding the presence of LMW glutenin-derived T cell stimulatory epitopes in breast milk, nothing is known. The assay specific for detection of the LMW-derived T cell stimulatory epitope of LMW glutenin (25) was not suitable for detection of this epitope in breast milk (this study).

Gliadin given to mothers on a gluten restricted diet, appeared in the breast milk between 2-4 hours after gliadin intake (31). Similar to our study, basal levels of gliadin were detectable even before intake of gliadin (31). From that study it is not clear however, whether the level of gliadin detected in the breast milk correlated with the gluten intake of the mothers. The basal level detected both in our study and in the study reported previously (31), might be explained by cross reactivity of the gluten specific antibodies used for detection and human proteins that have some similarity with gluten sequences.

For example, recently, cross reactivity between human anti-gliadin antibodies and a prolin glutamin rich neuronal protein, Synapsin I, has been described (33). Analyses of the human database for the minimal epitopes detected by our antibodies used for the

competition experiments, did not reveal any human proteins that might be recognized by our antibodies (result not shown). On the other hand, we did not have detailed

information on the strictness of the diet of mothers on a GFD and it is possible that the GFD diet was not kept strictly, either intended or unintended. Future experiments should be aimed at what proteins are detected by gluten specific assays in the breast milk of mothers on a strict GFD.

Upon ingestion, gluten proteins are digested by enzymes of the gastrointestinal tract including pepsin, trypsin, chymotrypsin and brush border enzymes. However, because of the high proline content of the gluten proteins, digestion is not complete and both of - gliadin (33-mer containing 6 overlapping T cell stimulatory epitopes including the Glia-α9 epitope detected in this study) and of -gliadin, degradation resistant peptides have been

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described (6,16). Those peptides contain the T cell stimulatory epitopes known to be involved in CD.

Moreover, in a recent study in which gluten degradation in the gastrointestinal system was mimicked in a gastrointestinal model, it was shown that also the HMW glutenin proteins are relatively resistant to degradation, probably even more resistant than gliadin proteins (26).

It has been suggested that the incomplete degradation of gluten in combination with the binding properties of the gluten peptides to HLA-DQ2 and HLA-DQ8 molecules, especially after deamidation by tTG, are the main cause of CD. Until now however, it is not known how those degradation resistant peptides cross the epithelial barrier and reach the lamina propria where, after endocytosis by dendritic cells, they are presented to the immune system. Analysis of the peptides detected in the breast milk in the near future might reveal in which form gluten crosses the epithelial barrier and enters the human body.

The presence of gliadin- and glutenin-derived peptides in breast milk of mothers indicates that infants are exposed to small amounts of gluten through the breast milk before the introduction of gluten into the infants’ diet, which normally is advised from the age of 6 months. It is tempting to assume that those low amounts of gluten peptides induce oral tolerance to gluten as is described for other antigens (34-36).

Lactating mammary glands are part of the integrated mucosal immune system and milk antibodies reflect antigenic stimulation of the mucosal immune system in the gut and in the airways. Secretory IgA from breast milk exhibits antibody specificities for an array of both intestinal and respiratory common pathogens (37) and dietary proteins like cows milk proteins and gluten (38,39). Until the infant develops its own secretory IgA and IgM producing B cell blasts and plasma cells, it is dependent on the maternal secretory antibodies present in breast milk.

Next to antibodies, other various dietary antigens are present in breast milk; however, dietary restriction during pregnancy and breastfeeding has shown no conclusive effect on the development of atopic diseases in the child (40,41). These antigens stimulate the maturation of the infants’ mucosal immune system (41) and under hyporeactive or immunosuppressive conditions, such as low antigen dose and/or presence of down regulatory cytokines as IL-10 and TGF- (42), activation of the immune system might be skewed towards a Th2 or tolerogenic phenotype.

CD is a disease with a strong genetic association. HLA-DR3, DQ2 positive individuals have a five times higher risk of developing CD than HLA-DR3, DQ2 heterozygous individuals and a more than 11 times higher risk than people with other HLA haplotypes (18). However, since most HLA-DR3, DQ2 positive individuals do not develop CD, it is generally assumed that gluten induces oral tolerance. Recently, dietary gluten specific, CD4+, IL-10 and TGF- producing regulatory T cells have been described. The cells are

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present in the mucosa and inhibit pathogenic gluten reactive T cells (43). It would be very interesting to know to which peptides those regulatory T cells respond and whether those peptides are present in breast milk and involved in the induction of oral tolerance against CD.

In conclusion, in the present study we show that low amounts of T cell stimulatory epitopes of gluten, both from gliadin and glutenin, are present in breast milk of mothers on a ND. Since oral tolerance to food antigens is induced early in childhood, in the period infants are breast-fed, these peptides might be involved in the induction of gluten tolerance. Future experiments should be aimed at the characterization and identification of the gluten peptides present in breast milk.

This knowledge will give some new insights in the way gluten tolerance is induced and will help us to generate novel strategies aimed at the prevention of the onset of CD.

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13. Arentz-Hansen H, McAdam SN, Molberg O, Fleckenstein B, Lundin KE, Jorgensen TJ, Jung G, Roepstorff P, Sollid LM. Celiac lesion T cells recognize epitopes that cluster in regions of gliadins rich in proline residues. Gastroenterology 2002;123:803-9.

14. Vader W, Kooy Y, van Veelen P, De Ru A, Harris D, Benckhuijsen W, Pena S, Mearin L, Drijfhout JW, Koning F. The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides. Gastroenterology 2002;122:1729- 37.

15. Nilsen EM, Lundin KE, Krajci P, Scott H, Sollid LM, Brandtzaeg P. Gluten specific, HLA-DQ restricted T cells from coeliac mucosa produce cytokines with Th1 or Th0 profile dominated by interferon gamma. Gut 1995;37:766-76.

16. Shan L, Qiao SW, Arentz-Hansen H, Molberg O, Gray GM, Sollid LM, Khosla C.

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17. Vader W, Stepniak D, Kooy Y, Mearin L, Thompson A, van Rood JJ, Spaenij L, Koning F.. The HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitude and breadth of gluten-specific T cell responses. Proc Natl Acad Sci U.S.A. 2003 Oct 14;100(21):12390-5. Epub 2003 Oct 6.

18. Mearin ML, Biemond I, Pena AS, Polanco I, Vazquez C, Schreuder GT, de Vries RR, van Rood JJ. HLA-DR phenotypes in Spanish coeliac children: their contribution to the understanding of the genetics of the disease. Gut 1983;24:532-7.

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27. Eckert R van. The PWG gliadin, a new reference material. Proceedings of the 16th meeting Working Group on Prolamin Analysis and Toxicity. Sitges, Spain, 2000.

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33. Alaedini A, Okamoto H, Briani C, Wollenberg K, Shill HA, Bushara KO, Sander HW, Green PH, Hallett M, Latov N. Immune cross-reactivity in celiac disease: anti-gliadin antibodies bind to neuronal synapsin I. J Immunol 2007;178:6590-5.

34. Friedman A, Weiner HL. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc Natl Acad Sci U.S.A 1994;91:6688-92.

35. Yoshida T, Hachimura S, Kaminogawa S. The oral administration of low-dose antigen induces activation followed by tolerization, while high-dose antigen induces tolerance without activation. Clin Immunol Immunopathol 1997;82:207-15.

36. Faria AM, Maron R, Ficker SM, Slavin AJ, Spahn T, Weiner HL. Oral tolerance induced by continuous feeding: enhanced up-regulation of transforming growth factor-

beta/interleukin-10 and suppression of experimental autoimmune encephalomyelitis. J Autoimmun 2003;20:135-45.

37. Goldman AS. The immune system of human milk: antimicrobial, antiinflammatory and immunomodulating properties. Pediatr Infect Dis J 1993;12:664-71.

38. Savilahti E, Tainio VM, Salmenpera L, Arjomaa P, Kallio M, Perheentupa J, Siimes MA.

Levels of IgA and cow milk antibodies in breast milk vs. the development of atopy in children. Low colostral IgA associated with cow milk allergy. Adv Exp Med Biol 1991;310:417-25.

39. Juto P, Holm S. Gliadin-specific and cow's milk protein-specific IgA in human milk. J

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40. Zeiger RS. Dietary aspects of food allergy prevention in infants and children. J Pediatr Gastroenterol Nutr 2000;30 Suppl:S77-S86.

41. Hoppu U, Kalliomaki M, Laiho K, Isolauri E. Breast milk--immunomodulatory signals against allergic diseases. Allergy 2001;56 Suppl 67:23-6.

42. Taylor A, Verhagen J, Blaser K, Akdis M, Akdis CA. Mechanisms of immune suppression by interleukin-10 and transforming growth factor-beta: the role of T regulatory cells. Immunology 2006;117:433-42.

43. Gianfrani C, Levings MK, Sartirana C, Mazzarella G, Barba G, Zanzi D, Camarca A, Iaquinto G, Giardullo N, Auricchio S, Troncone R, Roncarolo MG. Gliadin-specific type 1 regulatory T cells from the intestinal mucosa of treated celiac patients inhibit

pathogenic T cells. J Immunol 2006;177:4178-86.

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CHAPTER 3

Food questionnaire for assessment of infant gluten consumption

Erica G. Hopman, Jessica C. Kiefte- de Jong, Saskia le Cessie, Henriëtte A. Moll, Jacqueline C. Witteman, Sacha E. Bleeker, M. Luisa Mearin.

Clin Nutr 2007;26:264-71

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ABSTRACT

Background: In light of the possibly preventive role of timing and amount of gluten in celiac disease, it would be helpful to have a questionnaire to assess the gluten intake in infants.

Aims: Development and validation of a food questionnaire to assess gluten consumption in healthy infants aged 0-12 months (FQ-gluten).

Methods: A food frequency questionnaire, previously developed for the Generation R study, was adapted for the assessment of gluten intake. The results of a 2-day food record (FR) were compared with the results of this FQ-gluten.

Results: Eighty-seven parents filled in the FR and the FQ-gluten. The number of children who consume gluten and who are breast-fed is higher, reported in the FQ-gluten. The amount of gluten is comparable from the age of 3 up to 10 months, but at 11 and 12 months a higher gluten intake is reported using the FR, probably due to a larger variety of food products not detectable by the FQ-gluten. However, there is a high agreement in the food groups (Cohens' Kappa = 0.6-0.8).

Conclusions: This new, short, standardized, validated and easy to use FQ-gluten may be a useful instrument to assess gluten intake in infants, both at the individual and at the population level. The use of this method by investigators in other countries provides the opportunity for a better comparison of the results of gluten consumption in (co-

operative) studies throughout different countries.

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INTRODUCTION

Celiac disease (CD) is a lifelong disorder caused by intolerance for gluten, and CD is treated with a gluten-free diet. Breastfeeding (BF) and weaning influence the

development of the gastro-intestinal tract and it is possible that gradual introduction of antigens will lead to the development of oral tolerance (1-3). It is also likely that the response of the immune system to gluten is modified by BF (4,5).

The occurrence and disappearance of ‘epidemics’ of gluten intolerance after changes in Swedish infant feeding, during the 1980s and 1990s respectively, suggests that early feeding may be important in this respect. The analysis of the Swedish ‘experiment in nature’ has shown that ongoing BF during the period of gradual introduction of gluten- containing foods into the infant’s diet, significantly reduces the risk of gluten intolerance (6,7).

Several studies on gluten consumption and BF have been performed in different countries (7-13), but the methods used to assess gluten intake mostly were time

consuming and differed from each other. To our knowledge, there is no information on gluten introduction and gluten intake in the Netherlands.

As it is impossible to know precisely what a free living individual eats, there are various methods available for dietary assessment as an estimation of the intake, all of which have their advantages and disadvantages with regard to adequacy and work load (14,15). The food record (FR) assesses the intake recorded on the specific days on which the food and drinks are filled in; considering the intake on these days as a reflection of what someone normally eats. The food questionnaire (FQ) estimates how frequently certain foods are eaten during a specific period in time and only gives information on those foods or nutrients relevant to a specific question. Therefore, the FR may be the more accurate method, whereas the FQ may be the more representative, making it arguable which one is best to reflect true dietary intake (14). The FQ is an approach often used in

epidemiological studies and is less time consuming than the FR (15).

The aim of this study was to develop and validate an FQ for the assessment of gluten consumption (FQ-gluten) in children aged 0-12 months.

SUBJECTS AND METHODS Subjects

From February until July 2004, 192 consecutive parents of children aged 0-12 months who attended 4 Child Health Care Centres in the south-west part of the Netherlands were asked to participate in this study. In 2004 the Child Health Care Centres were attended by 91% of the infants in the Netherlands (16).

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Exclusion criteria were: 1) gestational age less then 36 weeks, 2) mental retardation or oral-motor dysfunction, 3) diagnosed food allergy, 4) impossibility of oral food intake, and 5) parents without enough knowledge of the Dutch language.

METHODS

Development of the FQ-gluten

As a basis for our new instrument to assess gluten consumption, we used the FQ

designed for a prospective cohort study in Rotterdam, the Netherlands; the Generation R study (17).

The Generation R study is a prospective population-based cohort study from foetal life until young adulthood. The study is designed to identify early environmental and genetic causes for growth, development and health in childhood and adulthood. For the Generation R study, 3 age specific FQs (0-2, 3-6 and 7-12 months of age) were developed by means of a standardized method in cooperation with the division of Human Nutrition and Epidemiology of the Wageningen University and Research Centre (18).

The FQs collect information on family characteristics and actual food intake, and, in retrospect, on the start and cessation of BF, the age at first introduction of food groups (e.g. fruits and meats), and on the intake of allergens and nutrients. However, they do not contain the whole spectrum of food products necessary to assess gluten intake.

In order to develop the FQ-gluten, we added gluten-containing food products according to the database of a recent food consumption study among young children aged 9 – 18 months (19) and according to the Dutch Food composition table (20), such as a variety of components for breakfast and the warm meal, flavoured milk products, ready-to-eat infant meals and porridges. The brand names of these products available in the Netherlands were derived from the list of food products (21). The FQ-gluten for children aged 7-12 months is the most extended FQ and comprises 68 items on food intake and BF (Table 1).

Table 1.Food frequency questionnaire for Dutch children at the age of 7 – 12 months of age.

1. This questionnaire is filled in by: □ Mother □ Father □ Both □ Other 2. Date of filling in the questionnaire ……../ ……../ ………

3. Date of birth of your child ……../ ……../ ………

4. Sex □ Boy / □ Girl

5. Order of birth □ 1st □ 2nd □ 3rd □ 4th or later

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6. Did you ever feed your child by breastfeeding □ No, go to question number 9 □ Yes

7. What age was your child when you stopped breastfeeding it? □ I still breast-feed □ Younger than 1 month □ Between 1 and 2 months □ Between 2 and 3 months □ Between 3 and 4 months □ Between 4 and 5 months □ Between 5 and 6 months □ Between 6 and 7 months □ Between 7 and 8 months □ Between 8 and 9 months □ Between 9 and 10 months □ Between 10 and 11 months □ Older than 11 months 8. How many times do you breast-feed at this moment? □ 1 – 2 times a day

□ 2 – 3 times a day □ 3 – 5 times a day □ 5 – 7 times a day □ More than 7 times a day 9. Do you feed your child formula feeding? □ No, go to question number 11

□ Yes

10. What kind of formula feeding do you give your child? □ Normal for the age □ For a younger age □ For an older age □ Adapted to food allergy 11. Do you feed your child porridge? □ No, go to question number 13

□ Yes, namely,

□ Bambix or Brinta or Molenaar □ Nutrix, Biobim junior □ Oatmeal porridge □ Semolina 12. How much porridge do you feed your child per day? □ If given by bottle:

□ Less than ½ bottle □ ½ - 1 bottle □ 1-1½ bottle □ 1½ - 2 bottles □ More than 2 bottles

□ If given by spoon:

□ Less than ½ plate □ ½ - 1 plate □ 1 – 1½ plate □ 1½ - 2 plates □ More than 2 plates 13. Do you add one of the following products to your child’s food (e.g. mixed with fruit, etc.)

□ Baby biscuits (Bambix, Liga 2^nd step, Liga 'big and strong'), rusk

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14. When you use ready-to-feed meals for your child, what brand do you normally use?

□ I do not use ready-to-feed meals □ Akwarius baby meals for 7 months □ Olvarit meal for 8, 15 or 18 months □ Olvarit 'Wereldreis' for 12 or 17 months □ Zonnatura meal for 8 or 12 months □ None of the above mentioned 15. When you use ready-to-feed fruit for your child what brand do you normally use?

□ I do not use ready-to-feed fruit □ Zonnatura for 8 or 12 months □ Olvarit fruit for 8 months with biscuit □ Zonnatura summer fruit with grains □ None of the above mentioned 16. With what frequency do you give your child the following food products?

Food product Never Less than

once a week

1-3 times

a week 4-6 times

a week Once

a day Twice

a day 3 times or more a

day Bread (1 slice=35 g)

French bread / Baguette (1 slice=15 g)

Currant bread (1 slice=35g) Rusk / crisp bread Honey-cake (1 slice=20g) Cracker/mazoth Multigrain rice waffle

Yoghurt or other milk product with flavour (150 ml)

Bambix or Brinta or Molenaar porridge (150ml)

Oatmeal porridge (150ml) Semolina porridge (150ml) Baby biscuit: Bambix, Liga 2^nd step, Liga 'big and strong'

“Lange vinger” biscuit Other sweet biscuits Soup stick Cake (1 slice=30 g) Pastry (1 piece=85 g) Ready-to-feed fruit Ready-to-feed warm meal Pasta: macaroni, spaghetti etc.

(1 portion = 50g)

Bulgur / couscous (1 portion=50g) Multigrain rice (1 portion= 50g) Pancake

Fritters (1 portion = 10) Pizza (1/8= 50g)

Crumbed products (meat, fish, chicken, cheese) (1 portion = 75g) Vegetarian burgers and balls (1 portion=75g)

Wheat flour based sauces (1 spoon=25g)

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17. Please note for the following products at what age you child first received them

Food product Never given < 3 months 3 - 6 months 6 - 9 months > 9 months Bread

French bread / baguette Currant bread

Rusk / crisp bread Honey-cake Cracker/mazoth Multigrain rice waffle

Yoghurt or other milk product with flavour

Bambix or Brinta or Molenaar porridge Oatmeal porridge

Semolina porridge

Baby biscuit: Bambix, Liga 2^nd step, Liga 'big and strong'

“Lange vinger” biscuit Other sweet biscuits Soup stick Cake Pastry

Ready-to-feed fruit Ready-to-feed warm meal Pasta: macaroni, spaghetti etc.

Bulgur Couscous Multigrain rice Pancake Fritters Pizza

Crumbed products (meat, fish, chicken, cheese)

Vegetarian burgers and balls Wheat flour based sauces

18. Does your child use medicine or vitamins at the moment? □ No □ Yes, namely:……….

Validation of the FQ-gluten

To validate the FQ-gluten, we asked the parents to fill in the new FQ-gluten and a 2-day FR (i.e. one weekday and one weekend day) of their child’s food intake in household measures and to precisely note the name of the manufacturer of the product used. A 2- day FR is an accepted method used in food consumption studies (19, 22).

Assessing gluten amount

We considered food products containing wheat, rye and barley as gluten-containing.

Since there is no information on the gluten content of food products, we used the method of Overbeek et al. (23) to calculate the content of gluten. Following this method, we multiplied the grams of gluten-containing protein according to the Dutch Food

Gluten intake and gluten-free diet in the Netherlands Hopman, G.D.

Gluten intake and gluten-free diet in the Netherlands

Gluten intake and gluten-free diet in the Netherlands

Contents

General introduction, aim and outline of this thesis

Presence of gluten proteins in breast milk:

implications for the development of celiac disease

Food questionnaire for assessment of infant gluten consumption